Spectroscopic testing methods that use one or more laser sources to illuminate samples are widely used in the field of microscopy. Depending on the type of measurement, the measuring technique in this case is subject to very different requirements.
For example, in SRS microscopy (stimulated Raman scattering; see for example Freudiger et al., Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy, Science 2008), two laser beams of different wavelengths are used. The two laser beams are guided towards the sample to be tested and are scattered thereon. During amplitude modulation (or, alternatively, polarization modulation or wavelength modulation, for example) of one of the two laser beams, a slight intensity fluctuation in the scattered light (a coherent, laser-like beam) is produced as a useful signal, which fluctuation has a high direct component, however. It is difficult to detect this intensity fluctuation (alternating component) involving a direct component.
The ratio of the direct component to the alternating component may be up to 10exp6, or the direct component of the light may be approximately one Watt, and this results in a significant amount of energy being introduced into the detector element (usually a photodiode). In addition to the increase in temperature due to the radiated light output, the direct component also results in a current flowing through the diode (which is usually reverse biased), and this also contributes to a significant increase in temperature. This has a negative effect on the measurement, in particular as a result of the operating point moving and the internal noise increasing.
The measurement is usually carried out by scanning, i.e. point-by-point raster scanning of the sample. This is usually carried out line by line, the laser being turned off when moving from one line to the next, which may result in an additional false signal when the laser is turned back on.